PROJECT SUMMARY Dendritic spines are the remarkable, highly specialized membrane compartments on neurons that house the postsynaptic, receiving end of many excitatory, glutamatergic synapses in the brain. They are highly plastic and change with learning, in development, and in disease. Dendritic spine and synapse changes have been linked to therapeutic responses to rapidly acting antidepressant drugs, but our understanding of the consequences of these treatments in specific neuronal classes are limited. This proposal relies on 2-photon multilaser assays to induce and evaluate the formation of new synapses, in order to measure how major depression and rapidly acting therapies regulate plasticity rules in specific neuronal groups. We will complement these focused assays with a new platform for genetically targeted proteomics in the cell classes implicated in major depressive disorder and its symptomatic amelioration by rapidly acting antidepressants. This work will provide the basis for a deep and broad understanding of synaptic and cellular changes induced by depressive state and rapidly acting antidepressant drugs. The unique synthesis of novel optical microscopy approaches, molecular tools, and proteomics chemistry represents a significant advance over canonical approaches to studying plasticity of neural circuits. The goal is to build a conceptual framework to enable harnessing the genesis of new synapses and the regulation of plasticity for mental health therapeutics. In addition, this work will help resolve fundamental mysteries surrounding the genesis of synapses and will develop useful tools for the neuroscience community.